Parietal cells are responsible for the secretion of hydrochloric acid in the stomach, using carefully modulated mechanisms to turn secretory activity on and off in coordination with normal dietary intake. The overall goal of this research is to establish the mechanism of parietal cell activation, that is, to discover how the parietal cell is transformed, structurally and functionally, from rest to full secretory activity. Cellular activation involves a cascade of biochemical events, often through protein phosphorylation. In the parietal cell these events effect the relocation of the acid pumping enzyme, the H,K-ATPase, from cytoplasmic vesicles to the apical plasma membrane where secretion occurs. This proposal consists of three major projects interrelated through their probing of how the parietal cell is regulated, and bearing on the more general question of how all cells are activated. One project will dissect the signaling events and identify mediators of cellular activation using intact and permeabilized models of gastric epithelial cells. The strategy is to develop simple in vitro systems to identify proteins that regulate transduction of secretory signals via their state of phosphorylation. The second major project examines the role of the actin cytoskeleton in membrane reorganization underlying HCl secretion. The research will ask whether there are essential changes in the dynamic assembly/disassembly of actin microfilaments associated with parietal cell activation, and seek to identify proteins that interact with the cytoskeleton to relay receptor- mediated signals for membrane remodeling and regulated secretion. Actin exists in several naturally occurring isoforms, which are minor structural variants of the same generic protein, but functional differences among actin isoforms have not been shown. Two isoforms, beta-actin and gamma- actin, are differentially localized within the parietal cell, thus making it an ideal system for asking the broad biological question of whether there is actin isoform specificity in functional activities, such as secretion, protein sorting and epithelial polarity. The third major project examines the structure and function of ezrin, a phosphoprotein that has been linked to membrane-cytoskeletal rearrangements during cellular activation and appears to be of special significance in parietal cell activation. Experiments will focus on phosphorylation of ezrin, identifying specific sites and pathways, and defining how phosphorylation alters the association of ezrin with actin and accessory proteins. Detailed structure of the ezrin molecule will also be established using X- ray crytallography for solving the atomic structure, and chemical and molecular manipulation to identify sites of ezrin-actin interaction and ezrin-membrane association.
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